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[
International Worm Meeting,
2021]
The genome size variation is an "old" question in evolutionary biology. However, its causes and consequences are still a debate. There are various intraspecies models to study genome size variations, which usually hold very limited size change between pairs. As a result, sophisticated and pricy detection methods are required to monitor the change of genome size. And the impact of large size variation can not be tested. The inter-species models, however, could have significant genome size variation. One close sister pair among Caenorhabditis clade, the C. briggsae and C. nigoni, has around 30% genome size difference. They can cross with each other and produce fertile females, and can be used to study genome size variation and hybrid incompatibility. One major disadvantage of inter-species model is that, the F1 male is either dead for sterile, which hinder the monitoring of further offspring by F1 crossing. We generated a lot of introgression strains with C. nigoni genome background with a small fragment from C. briggsae genome. By incorporating different C. briggsae X chromosome fragments into C. nigoni background, we are able to produce a homozygotic introgression strain (ZZY10253), which could mate with C. briggsae male and produce both fertile F1 females and males. By breeding the F1 worms with 10 x 10 crossing, we can monitor the competition of the two haplotypes, which has 30% size difference, by checking the ratio of two haplotypes using NGS sequencing. We have sequenced some F7 and F20 lines. And the long haplotype, the C. nigoni haplotype, has become dominant (78%) in F7 populations, especially in the X chromosome (92%). With this speed, we expected to see a 100% recovery of ZZY10253 genotype after F15. But to our surprise, the F20 lines still maintained at least 8% of C. briggsae haplotype, and all these lines (n=8) have two autosomes remained. These remained autosomes may reflect an interaction between the X introgression fragment and autosomes, which grant these individuals some advantage in growing.
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[
Cell Host Microbe,
2009]
Similarities in innate immune signaling exist between mammals and the nematode Caenorhabditis elegans. Now, Ziegler et al. (2009) and Ren et al. (2009) demonstrate that a protein kinase C delta homolog in C. elegans is involved in innate immunity, providing evidence that the conservation of immune signaling networks extends further than previously thought.
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[
International Worm Meeting,
2017]
Differential expression of orthologous genes could be a product of divergence in cis-regulatory elements and/or trans-factors. Analysis of transcriptomes in F1 hybrids between closely related species provides an opportunity for systematically dissecting the roles of cis-elements and the trans-factors in controlling allele-specific expression. This analysis could also provide insight into the mechanism controlling genome stability, for example, through transposon silencing. The F1 hybrid transcriptomes have been analyzed in most model organisms, but never been examined in nematode species. Here we perform transcriptome analyses in the F1 female hybrids from reciprocal crosses between C. briggsae and C. nigoni with their parental hermaphrodites/females as a control. The reciprocal transcriptomes demonstrate a nearly perfect correlation between each other, indicating few genomic imprinting events in the parents. Approximately 8,000 orthologous pairs have at least 10 sequencing reads which are used in the subsequent analysis. Over 5,000 of them demonstrate differential allelic expressions in the hybrids, which mirror their expression divergences in their respective parents, indicating that cis-regulation plays a major role in controlling expression of these pairs in the hybrids. Roughly another 800 pairs show a significant averaging effect of their parental expression divergences in the hybrids, supporting the allelic expression in the hybrids is predominately regulated by trans-factors. The remaining 2,000 pairs demonstrate conserved expression in both parents and their hybrids. Unlike the F1 hybrids in Drosophila species in which a sharp increase in transposon expression is frequently observed, we barely see any significant increase in the expression of the mobile elements, suggesting compatible endogenous RNAi pathways between the two species in transposon silencing. Taken together, we demonstrate a lack of parent-of-origin differential allelic expression in female reciprocal F1 hybrids between C. briggsae and C. nigoni, in which allele-specific expressions of orthologous pairs are mostly controlled by cis-regulatory elements. Transposon-silencing mechanisms appear to be conserved between the two species.
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[
International Worm Meeting,
2013]
HI serves as reproductive barrier limiting gene flow between different species. Characterization of HI remains the key to understanding of speciation. Inter-species HI loci have been isolated primarily between model organisms and its related species. However, C. elegans has been prevented from isolation of inter-species HI loci due to lack of a sister species with which it can mate and produce viable progeny. To circumvent the limitation, we set to isolate HI loci between C. briggsae and its sister species, C. sp. 9 which has recently been isolated. We first generated 80 stable transgenic strains expressing GFP markers in C. briggsae. We next mapped the GFP integration sites through repeated back-crossings/introgression into C. sp.9 followed by PCR genotyping. The markers demonstrate roughly even distribution over C. briggsae genome. We then rendered the introgression fragment homozygous if possible in an otherwise C. sp.9 background. We were able to produce a total of nine viable lines carrying independent homozygous introgression fragments with four on autosome and five on X chromosome. The remaining introgression fragments seem inviable as homozygote. The sizes of introgression fragment range from 0.5 Mb to as big as 10 Mb. We characterized the HI for all the homozygous introgressions as well as for 17 other heterozygous introgressions, representing nearly 80% of C. briggsae genome. Surprisingly, most of the homozygous introgressions on autosome dramatically reduce viabilities while those on X chromosome demonstrate modest incompatibilities, inconsistent with large X theory. Strikingly, none of the homozygous introgression lines significantly affects male sterility or viability though the Haldane's rule is widely obeyed during the initial introgression. Nevertheless, we did observe that introgressions leading to male sterilities are mainly located in the middle of X chromosome and mapped male viability loci onto an approximately 1Mb region of X chromosome. In addition, we found that the HI loci are largely recessive. Our work build a framework that enables nematode species to be used as a model for investigation of the mechanisms of inter-species HI that has so far not been successful.
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Li, Runsheng, Xie, Dongying, Ding, Qiutao, Ren, Xiaoliang, Zhao, Zhongying, Bi, Yu
[
International Worm Meeting,
2019]
High throughput RNA-seq using cDNA has played a key role in delineating transcriptome complexity, including alternative transcription initiation, splicing, polyadenylation and base modification. However, the reads derived from current RNA-seq technologies are usually short and deprived of information on modification during reverse transcription, compromising their potential in defining transcriptome complexity. Here we applied direct RNA sequencing with ultra-long reads from Oxford Nanopore Technologies (ONT) to study the transcriptome complexity in C. elegans. We sequenced native poly-A tailed mRNAs by generating approximately six million reads from embryo, L1 larvae and young adult animals, with average read lengths ranging from 900 to 1,100 bps across stages. Around half of the reads represent full-length transcripts judged by the presence of a splicing-leader or their full coverage of an existing transcript. To take advantage of the full-length transcripts in defining transcriptome complexity, we devised a novel algorithm to predict and quantify the isoforms using read's mapping tracks rather than established intron/exon structures. We recovered at least 26,000 out of the existing 33,500 isoforms, and identified roughly 56,000 novel ones. Intriguingly, stage-specific expression at the level of gene and isoform demonstrates little correlation. Finally, we observed an elevated level of modification in all bases in the coding region than the UTR. Taken together, the native long reads by ONT are expected to deliver new insights into RNA processing and modification and their underlying biology.
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Xie, Dongying, Bi, Yu, Li, Runsheng, Zhao, Zhongying, Yan, Cheung, Shao, Jiaofang, Ren, Xiaoliang
[
International Worm Meeting,
2015]
Genetic basis of hybrid incompatibility (HI), a collective term of compromised fitness in hybrid, including sterility and lethality, has been intensively studied in Drosophila species, but remains poorly understood in other species, especially in nematodes. Recent discovery of a C. briggsae sister species, C. nigoni, has opened up the possibility of dissecting the genetic basis of HI in nematode species. We have generated 96 chromosomally integrated GFP markers in the C. briggsae genome and mapped them into the defined locations by PCR and Next-Generation Sequencing (NGS). Aided by the marker, we backcrossed the GFP-associated C. briggsae genomic fragments into C. nigoni for at least 15 generations and produced 111 independent introgression lines. The introgression fragments cover most of the C. briggsae genome. We finally dissected the patterns of HI by scoring the embryonic lethality, larval arrest, sex ratio and male sterility for each introgression line, through which we identified pervasive HI loci and produced a genome-wide landscape of HI between the two nematode species, the first of its type for any non-Drosophila species.To further understand the genetic basis of HI between the two species, we focused on the molecular understanding of two introgression fragments that produce male sterility but are located on different parts of X chromosome. To this end, we performed RNA-seq for both sterile and parental males and quantified the reads using C. briggsae genome as a reference. A total of 957 genes were found differentially expressed (Fold Change>1.5, FDR<0.05) in both hybrids compared with both parental species with most genes showing down-regulation. Surprisingly, 364 out of 957 genes are those enriched during spermatogenesis, suggesting that preferential down-regulation of spermatogenesis genes caused by the introgression may at least partially to be blamed for the observed sterility. More details on the differentially expressed genes will be presented in the meeting.
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Zhao, Zhongying, Li, Runsheng, Young, Amanda, Zhang, Zhihong, Ren, Xiaoliang, Hsieh, Chia-Ling
[
International Worm Meeting,
2015]
Next-Generation Sequencing permits rapid acquisition of high-throughput DNA sequences, but its reads are relatively short in length, limiting their use in genome-based studies. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking, especially in resolving the repetitive sequences. We evaluate the promise and deficiency of the long reads in these aspects using C. elegans genome. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences that extend over certain length prevents pre-assembly of the long reads inside this genomic region. Second, the reads can reliably recover the missing but not the extra sequences in C. elegans genome. 24X of the long reads allow the recovery of at least 40 Kb of missing genomic DNAs that are located inside or outside coding region. Finally, an N50 size of at least 86 Kb can be achieved for the contigs that are de novo assembled with the 24X reads. However, substantial mis-assembly errors are observed which are caused by either mis-assembled long read or flanking repetitive sequences, highlighting a need for novel assembly algorithm to accommodate the long reads in de novo genome assembly. The long reads are expected to be useful in generating a "finished-grade" genome of other nematode species if coupled with independent data such as those from PacBio or mate-pair sequencing or repairing the existing nematode draft genomes on its own.
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Xie, Dongying, Bi, Yu, Shao, Jiaofang, Yan, Cheung, Ren, Xiaoliang, Zhao, Zhongying
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Systematic characterization of hybrid incompatibility (HI) between related species remains the key to understanding speciation. The genetic basis of HI has been intensively studied in Drosophila species, but remains largely unknown in other species, including nematodes. This is mainly due to the lack of a sister species with which C. elegans can mate and produce viable progeny. Recent discovery of a C. briggsae sister species, C. sp.9, opened up the possibility of dissecting the genetic basis of HI in nematode species. However, lack of dominant and visible markers prevent efficient mapping of HI loci between the nematode species. To systematically isolate HI loci between the two nematode species, we first generated 96 chromosomally integrated GFP markers in C. briggsae genome and mapped them into the defined locations by PCR and Next Generation Sequencing (NGS). High density of the dominant and visible markers permits repeated backcrossing of the GFP linked C. briggsae genomic fragment into the C. sp.9 background, allowing definitive assignment of an HI phenotype into a specific genomic interval. We next backcrossed the individual markers into C. sp.9 for at least 15 generations and produced 111 independent introgression lines, which cover most of the C. briggsae genome. We finally dissected the HI patterns by scoring embryonic lethality, larval arrest, sex ratio, fertility, male sterility and inviability for a subset of 36 introgression lines and identified pervasive HIs, providing a genome-wide landscape of HI between nematode species for the first time. Observed HI phenotypes are consistent with the Haldane's rule and the fertility data from homozygous introgressions supports the rule of large X effect. The large collection of introgression lines provides an invaluable resource for further characterization of the genetic and molecular mechanisms of HI in nematode species, allowing comparative analysis of speciation genetics between nematodes and other species.
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Ho, Vincy, Xie, Dongying, Ding, Qiutao, Chan, Lu-Yan, Li, Runsheng, Ren, Xiaoliang, Zhao, Zhongying, Ye, Pohao
[
International Worm Meeting,
2021]
Ribosomal genes (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosome. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. Here, we generate ultra-long Nanopore and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform manual assembly of the cluster using the long reads that carry these units, which led to an assembly of rDNA cluster consisting of up to 167 5S rDNA units. The ordering and copy number of various rDNA units are indicative of separation time between strains. Surprisingly, we observed a drastically lower level of variation in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than C. elegans N2 strain, suggesting a unique mechanism in maintaining the rDNA cluster stability in the N2. Single-copy transgenes landed into the rDNA cluster shows the expected expression in the soma, supporting that rDNA genomic environment is transcriptionally compatible with RNA polymerase II. Delineating the structure and variation of rDNA cluster paves the way for its functional and evolutionary studies.
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Stamatoyannopoulos, John, Ho, Vincy, He, Kan, Shao, Jiaofang, An, Xiaomeng, Zhao, Zhongying, Ren, Xiaoliang, Xie, Dongying, Wong, Ming-Kin, Wang, Hao, Yan, Bin
[
International Worm Meeting,
2013]
Cell fate specification demands a hierarchy of regulatory events. Initial specification is typically achieved by a master regulator which is relayed by tissue-specific regulatory proteins usually transcription factors for further enforcement of cell identities, but how the factors are coordinated between each other to "finish up" the specification remains poorly understood. C. elegans epidermis specification is initiated by a master regulator ELT-1 which subsequently activates its targets NHR-25 and ELT-3, two epidermis specific transcription factors, thus providing a superior paradigm for illustrating how the tissue specific regulatory proteins work together to enforce cell fate specification. Here we addressed the question through contrasting genome-wide in vivo binding targets between NHR-25 and ELT-3 that are important for epidermis development but not required for its initial specification in C. elegans. We first identified in vivo binding targets of NHR-25 by ChIP-seq and then compared them with those of ELT-3, the result of which demonstrated apparently differential regulation of metabolism but coordinated control of epidermal development between the two. Functional validation of the targets demonstrated that both activating and inhibitory roles of NHR-25 in regulating its targets. We further showed differential regulation of specification of AB and C lineage derived epidermis. Our results provide insights into how tissue specific regulatory proteins coordinate with one another to enforce cell fate specification initiated by its master regulator. Combined with functional analysis, we assembled a comprehensive gene network underlying C. elegans epidermis development and physiology which are likely to be broadly used across species.